The present invention relates to a scanning confocal microscope for increasing the processing speed by improving scanning control of focused light with respect to a sample and data processing.
A scanning confocal microscope utilizes a confocal effect of illuminating the surface of a sample to be observed (to be referred to as a sample hereinafter) with a light spot from a point source, focusing light transmitted through or reflected by the illuminated sample surface into a light spot again to form an image on a detector having a pinhole, and obtaining luminance information of the formed image from the detector.
A general scanning confocal microscope will be described with reference to a schematic view shown in FIG. 1.
A light spot emitted by a point source 1 passes through a half-mirror 2, and forms a spot on the surface of a sample 4 via an aberration-corrected objective lens 3.
Light reflected by the spot-illuminated sample 4 passes through the objective lens 3 again, and is reflected by the half-mirror 2 and focused. A pinhole 5 is formed at the focal position. The reflected light having passed through the pinhole 5 is detected by a photodetector 6.
The scanning confocal microscope can raster-scan the sample 4 with such a light spot to two-dimensionally scan the entire measurement region of the surface of the sample 4, and display as an image a detection signal of the reflected light obtained by the photodetector 6, thereby obtaining a two-dimensional image of the surface of the sample 4.
This two-dimensional scanning adopts, e.g., a galvanoscanner or resonant scanner for the X direction and a galvanoscanner for the Y direction.
When the X- and Y-scanners are a combination of galvanoscanners, the scanning rate of the surface of the sample 4 is about 1 image/sec. When the X-scanner uses a resonant scanner in order to increase the scanning rate, the scanning rate is about 5 images/sec. Note that the number of pixels is 1,024xc3x97768.
Such a scanning confocal microscope realizes scanning capable of obtaining an in-focus image of the entire surface of a stepped sample 4 by the above-described confocal effect (to be referred to as extended scanning).
This scanning utilizes the fact that the luminance of the sample 4 maximizes at an in-focus position. Luminance information of the sample 4 obtained at the position of the objective lens 3 (or sample 4) is compared with that of the sample 4 obtained at a position of the objective lens 3 (or sample 4) slightly shifted to the optical axis.
By leaving a pixel having a higher luminance between identical pixels of the two images, an image of the sample 4 finally obtained within a given range along the optical axis becomes an in-focus two-dimensional image of the entire surface of the sample 4.
When the luminance is determined to be higher in pixel comparison, a position along the optical axis at that time can be stored to finally attain height (three-dimensional) information of the sample 4.
In extended scanning, however, information about the height of the sample 4 or the like can be obtained only after the sample 4 is two-dimensionally scanned and the objective lens 3 (or sample 4) is moved along the optical axis within a given range. The acquisition time is determined by the scanning rate, the data processing speed, the moving time of the objective lens 3 (or sample 4), and the entire moving range. The time is much longer than the time necessary for acquiring only general two-dimensional information.
To shorten the total measurement time when one measurement operation requires a large amount of image data (luminance information), like extended scanning, the acquisition time (image update time) of luminance information of the sample 4 by two-dimensional scanning must be shortened.
When many regularly aligned stepped samples are to be successively measured, like recent bump measurement, the measurement time of the sample 4 must be shortened.
Techniques disclosed to increase the scanning rate include, e.g., two-dimensional scanning using an acoustooptical element. According to this technique, the scanning angle cannot be set large, so the entire field of view is limited.
In addition, two-dimensional scanning using a CCD line sensor cannot realize a resolution which can be obtained by a general confocal optical system.
It is, therefore, an object of the present invention to provide a scanning confocal microscope capable of measuring a necessary region at a high speed regardless of the scanning means, magnification (observation field of view), and resolution in measuring a sample.
In extended scanning, after an image is captured at a certain focal position by two-dimensional scanning, the objective lens 3 (or sample 4) is moved to the next focal position along the Z-axis at a predetermined pitch to capture the next image by two-dimensional scanning.
In general, the image capture period of two-dimensional scanning at the focal position is not synchronized with the focal position moving time. Before movement of the focal position is completed during the blanking period of scanning, image capture by two-dimensional scanning starts, and the captured image becomes wasteful data.
For this reason, the next image capture must be devoted to an idle (no-data acquisition) time. Acquiring a desired number of images requires a long time.
To shorten the image capture time, some microscopes execute focal movement of the objective lens 3 (or sample 4) while capturing images by two-dimensional scanning. In these microscopes, the focal position moves during image capture scanning, so that an image is captured by obliquely scanning a sample. Considering the process in which height information is obtained by extended scanning, no accurate information can be obtained.
The present invention has been made in consideration of the above situation, and has as its object to provide a scanning confocal microscope capable of acquiring accurate image information within a short time.
According to the first aspect of the present invention, a scanning confocal microscope is characterized by comprising scanning range designation means for designating a two-dimensional scanning range of focused light with respect to a sample, two-dimensional scanning means for two-dimensionally scanning the sample with the focused light within the two-dimensional scanning range designated by the scanning range designation means, light-receiving means for receiving one of light reflected by the sample, fluorescence, and transmitted light of the focused light two-dimensionally scanned by the two-dimensional scanning means, and image acquisition means for acquiring an image of the sample on the basis of one of the reflected light, the fluorescence, and the transmitted light that is received by the light-receiving means.
According to the second aspect of the present invention, a scanning confocal microscope is characterized by comprising two-dimensional scanning means for two-dimensionally scanning a sample with focused light within a two-dimensional scanning range, light-receiving means for receiving one of light reflected by the sample, fluorescence, and transmitted light of the focused light two-dimensionally scanned by the two-dimensional scanning means, data acquisition range designation means for designating a data acquisition range on image data of the sample obtained based on one of the reflected light, the fluorescence, and the transmitted light that is received by the light-receiving means, and image acquisition means for acquiring an image of the sample on the basis of image data within the data acquisition range designated by the data acquisition range designation means out of the image data of the sample obtained based on one of the reflected light, the fluorescence, and the transmitted light that is received by the light-receiving means.
According to the third aspect of the present invention, a scanning confocal microscope is characterized by comprising scanning range designation means for designating a two-dimensional scanning range of focused light with respect to a sample, two-dimensional scanning means for two-dimensionally scanning the sample with the focused light within the two-dimensional scanning range designated by the scanning range designation means, light-receiving means for receiving one of light reflected by the sample, fluorescence, and transmitted light of the focused light two-dimensionally scanned by the two-dimensional scanning means, data acquisition range designation means for designating a data acquisition range on image data of the sample obtained based on one of the reflected light, the fluorescence, and the transmitted light that is received by the light-receiving means, and image acquisition means for acquiring an image of the sample on the basis of image data within the data acquisition range designated by the data acquisition range designation means out of the image data of the sample obtained based on one of the reflected light, the fluorescence, and the transmitted light that is received by the light-receiving means.
According to the fourth aspect of the present invention, a scanning confocal microscope is characterized by comprising two-dimensional scanning means for two-dimensionally scanning a sample with focused light, focal position adjustment means for adjusting a focal position of the focused light with respect to the sample, and control means for inhibiting two-dimensional scanning of the two-dimensional scanning means during a focal position adjustment period of the focal position adjustment means.
According to the fifth aspect, the fourth aspect is characterized in that the control means manages a horizontal sync signal output every 1-line scanning of the two-dimensional scanning means and a vertical sync signal output every frame scanning, instructs the focal position adjustment means to start adjusting the focal position and inhibits two-dimensional scanning of the two-dimensional scanning means in response to the vertical sync signal, and starts two-dimensional scanning of the two-dimensional scanning means after the focal position adjustment means adjusts the focal position.
According to the sixth aspect, the fifth aspect is characterized in that the control means further manages a control signal output subsequently to the vertical sync signal of the two-dimensional scanning means, when end of adjustment of the focal position by the focal position adjustment means is informed before the control signal, does not inhibit two-dimensional scanning of the two-dimensional scanning means, when end of adjustment of the focal position by the focal position adjustment means is informed after the control signal, temporarily inhibits two-dimensional scanning of the two-dimensional scanning means upon generation of the control signal, and starts two-dimensional scanning of the two-dimensional scanning means after end of adjustment of the focal position.
As a result, according to the present invention, two-dimensional scanning by the two-dimensional scanning means and movement of the focal position by the focal position adjustment means in extended scanning can be synchronized to eliminate a wasteful two-dimensional scanning period (idle period during which no data can be acquired).
Also, according to the present invention, the period between the vertical sync signal of the two-dimensional scanning means and a successively output control signal can be used for adjustment of the focal position by the focal position adjustment means.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.